PROTAC for Bruton’s tyrosine kinase degradation alleviates inflammation in autoimmune diseases

DMSO/Ibrutinib/L18I (100 nM) treatment and various stimuli processing time patterns


BM12-induced lupus-like mouse model
To induce lupus-like model, splenocytes (1 x 10 7 per mouse) from age and gender-matched BM12 mice were adoptively transferred to 6-8-week wild-type C57BL/6 mice for two times on a weekly basis.Vehicle, Ibrutinib and L18I were administered daily from weeks 2 to 4, and the mice were sacrificed at week 4 for detection of autoantibodies and glomerular immune-complex deposition.

Enzyme-linked immunosorbent assay (ELISA)
For anti-dsDNA detection, 4 μg/mL dsDNA was coated on the ELISA plates with DNA-coating solution (Thermo Fisher) overnight at 4 ℃.Blocked with 5% skim milk for 2 h at room temperature (RT), these plates were added gradient dilutions of mouse serum and incubated for 1.5 h at RT. Peroxidase-conjugated goat anti-mouse IgM and IgG were used for detection.These plates were washed with PBS/0.05%Tween-20 between each two steps.To detect the total IgM in pristaneinduced DAH mice, 4 μg/mL goat anti-mouse IgM was coated on the ELISA plates overnight at determined by the maximum dilution that is close to the two times of background OD value.Antinuclear antibodies (ANA) were quantified through immunofluorescence with ANA detection kit following the recommended protocols.

DAH induction and identification of DAH degree
DAH was induced by a one-time intraperitoneal injection of 0.8 ml pristane (MACKLIN) in 6-8 week wild-type C57BL/6 mice.We identified the degree of DAH after 2 weeks of pristane induction.

L18I and Ibrutinib administration
For mouse experiments, L18I and Ibrutinib were dissolved in PBS containing 10% DMSO and 10% Cremophor EL.The vehicle group was treated with PBS containing 10% DMSO and 10% Cremophor EL.We set the healthy and vehicle group as negative and positive control, respectively.
Vehicle, Ibrutinib and L18I (50 mg/kg, i.p., twice a day) were administered daily from weeks 2 to 4 after BM12-splenocytes transfer.The drugs were administered daily (50 mg/kg, i.p., once or twice a day) from 2 days to 14 days after pristane injection.For in vitro cell experiments, L18I and Ibrutinib were dissolved in DMSO and cells were treated with 100 nM or 200 nM L18I and Ibrutinib under different experimental conditions.

Western Blotting
To detect the off-target effects of L18I, Jurkat cells, PC-9 cells and WM115 cells were treated with L18I for 24 hours.To detect the activation of signaling pathways, Ramos cells were treated with Ibrutinib or L18I in presence or absence of LPS stimulation for 24 hours.To detect the BTK expression and degradation within different immune cell lines including Ramos, THP-1, Jurkat, U266 and J558L cells, these cells were treated with different concentrations of L18I for 24 hours.
The collected cells were lysed using RIPA buffer containing protease/phosphatase inhibitor cocktail (Cat No. 04693124001, Roche, Mannhein, Baden-Wuerttemberg, Germany).The BCA assay was carried out to quantify protein (Cat No. 23227, Thermo), and equivalent amounts of protein samples were separated by SDS-PAGE contained 4-12% acrylamide (Cat No. NPO335BOX, Invitrogen, Carlsbad, CA).After the protein was transferred to a PVDF membrane, the proper primary and secondary antibodies were probed and used for chemiluminescence detection.To detect the BTK degradation in mice and different primary cells, Vehicle and L18I (50 mg/kg, i.p., twice a day) were administered daily for 4 days.Different tissues were harvested from animals and flash frozen in liquid nitrogen.Specifically, small pieces (10-20 mg) of the collected tissues were lysed with RIPA lysis buffer containing 1% PMSF and 1% protease inhibitor on ice for 1 h.Primary mouse monocytes, T cells, and B cells were purified from peripheral blood and spleen, and were lysed using RIPA buffer containing protease/phosphatase inhibitor cocktail.Protein samples were collected for concentration determination and further analysis as described previously

Flow cytometry of lung immune cells
Fresh lungs were obtained and chopped with scissors.The digestion solution was prepared by adding 0.5 mg/mL collagenase IV and 0.1 mg/mL DNase I to the incomplete RPMI medium.Each lung was digested with 5 ml of digestion solution on a shaker at 37 ℃ for 1.5 h, gently pressed with a syringe rubber plug, and then passed through a 70 μm screen.The digested cells were collected, washed once with PBS and lysed with red blood lying buffer, and the lung single-cell suspension was obtained.After blocking with 2.4G2 on ice for 15 min, the cells were stained with CD11b, Ly6G, Ly6C, CD11c, Siglec-F, MHCII and B220 antibodies for 20 min at 4℃, and washed once with PBS.Then the dead and alive cells were distinguished by 7-AAD antibody, and the cells were directly detected by Fortessa flow cytometer (BD) by adding appropriate amount of PBS.In order to detect the levels of BTK protein, cells were fixed and permeabilized using Fix/Perm kit for intracellular staining of BTK antibody, and the fluorescence intensity of BTK was detected by Fortessa flow cytometer (BD).We use the MFI of BTK in the NC group without BTK antibody as the background.The BTK protein content was characterized by subtracting the background MFI value from the BTK-MFI of vehicle group and L18I group, respectively.Then, with the average value of vehicle group normalized as 1, the relative amount of BTK in each mouse was calculated, and the degradation ratio of BTK protein could be calculated.

Stimulation of primary B cells in vitro
Primary B cells were isolated from murine spleen and the purity of B cells was confirmed by flow cytometry.2 x 10 5 cells were cultured in 96-well plates, treated with DMSO, Ibrutinib and L18I (100 or 200 nM) and then stimulated by F(ab')2 anti-mouse IgM (10 g/mL) and LPS (1 g/mL).
After 24 hours of stimulation, the expression of activation makers CD25, CD69 and CD86 on the surface of primary B cells was analyzed by flow cytometry.

RNA-seq analysis
1 x 10 6 primary B cells were cultured in 24-well plates, treated with the DMSO, Ibrutinib and L18I (200 nM) and then stimulated by LPS (1 g/mL) for 12 hours, with the control group without LPS stimulation.Then total RNA was purified by trizol method, and the RNA integrity was confirmed by 2100 Bioanalyzer (Agilent Technologies Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen).Sequencing was performed by Illumina NovaSeq sequencer (Illumina, San Diego, CA, USA) according to the manufacturer's instructions, and data processing and analyzing were performed by CapitalBio Technology (Beijing, China).

Stimulation of FcγR signaling, TLR signaling and NLRP3 inflammasome
To stimulate the FcγR signaling of BMDMs, 1 g/mL mouse IgG was incubated in a 24-well plate at 4 ℃ for 24 h.The induced BMDMs were digested and counted, pretreated with DMSO, Ibrutinib (100 nM) or L18I (100 nM) and placed in the well coated with mouse IgG for 24 h.To stimulate the TLR signaling, BMDMs were pretreated with DMSO, Ibrutinib or L18I for 4 hours and then stimulated by 0.5 g/mL LPS for 20 h.BMDMs or THP1 cells, pretreated with DMSO, Ibrutinib or L18I for 2 h, were primed with 50 ng/mL, 100 ng/mL or 200 ng/mL LPS for 3 h at 37℃ and then stimulated with 10 M Nigericin (NI) for 1h.After stimulation, cells were collected to quantify the Tnfa, Il1b and Il6 expression, and supernatants were collected to determine the IL-1β levels by ELISA using human or mouse IL-1β detection kit.

RT-qPCR
Total RNA of stimulated cells was obtained by RNA extraction kit, and was reversed into cDNA by reverse transcription kit.qPCR was performed using SYBR in Bio-Rad CFX96 AGCCTCCGACTTGTGAAGTGGT). Gene expression was normalized to mouse-Actin (forward: GTGACGTTGACATCCGTAAAGA; reverse: GCCGGACTCATCGTACTCC), and was determined by using the ΔΔCt method.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 5.0 software and displayed in the form of mean ± SD.Flow cytometry analysis was performed with Flowjo software.Images were processed and analyzed using Image J software.*P < 0.05, **P < 0.01, ns: no significance.